A comparison of levels of glutathione transferases, cytochromes P450 and acetyltransferases in human livers.
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Acetyltransferases↗
Biomedical subjects
B Ketterer
Publications and source records attributed to B Ketterer.
The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris.
1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.
Amino Acid Sequence↗
Glutathione S-transferase mu locus: use of genotyping and phenotyping assays to assess association with lung cancer susceptibility.
In mammals, the cytosolic glutathione S-transferases (GSTs; EC 2.5.1.18) are a supergene family comprised of four multigene families, named alpha, mu, pi and theta. In man, within the mu class gene family there is a gene (the GSTmu 1 locus) that is polymorphic and is only expressed in 50-55% of individuals. It has previously been reported, using trans-stilbene oxide (tSBO) as a specific substrate for the expressed phenotype, that smokers with the null phenotype had a greater susceptibility to lung cancer. In a subsequent study, it was shown that on Southern blot analyses of human DNAs using a GSTmu 1 cDNA probe a DNA fragment was absent in certain individuals. The absence of this band correlated with the tSBO null phenotype. In the present work, DNA clones derived from GST mu class genomic sequences were used as probes in Southern blot analyses and confirmed the correlation between the lack of a DNA fragment and the null phenotype; moreover in this case, using radioimmunoassay for the GST mu protein, these probes were then used in a genotyping assay to investigate further the association of GSTmu 1 polymorphism with susceptibility to lung cancer. It was found that in a control group of 225 individuals, of unknown smoking history, 42% lacked the restriction fragment and were homozygous null, and therefore 58% were either heterozygous or were homozygous normal. Among 228 lung cancer patients, which included all tumour types, a similar distribution occurred, namely 43% were homozygous and 57% were heterozygous or homozygous normal. If, however, the tumours were analysed by tumour type a small but significant positive correlation with the homozygous null genotype was seen in squamous carcinoma of the lung, and an apparently negative correlation with adenocarcinoma of the lung.
Blotting, Southern↗
Selectivity of rat and human glutathione S-transferases in activation of ethylene dibromide by glutathione conjugation and DNA binding and induction of unscheduled DNA synthesis in human hepatocytes.
The major DNA adduct formed by the carcinogen ethylene dibromide (EDB) is S-[2-(N7-guanyl)ethyl]glutathione. This adduct results from the glutathione S-transferase (GST)-catalyzed conjugation of EDB with glutathione (GSH), which generates an episulfonium ion capable of reacting with cellular nucleophiles. Purified rat and human GST enzymes were compared for their ability to conjugate EDB with GSH and displayed high selectivity. Of the six forms of rat GST tested, conjugation was catalyzed by the alpha class enzyme 2-2 and, to a lesser extent, by the mu class enzyme 3-3. Of the three classes of cytosolic human GST, EDB conjugation was catalyzed by the alpha class enzymes. Three dimers of the human alpha class (alpha x-alpha x, alpha x-alpha y, and alpha y-alpha y) were separated by chromatofocusing. The alpha x-alpha x preparation demonstrated the highest specific activity. Rat microsomal GST had negligible activity for the conjugation of EDB with GSH. The levels of EDB-DNA adducts formed in rat and human hepatocytes were compared. DNA was isolated from both rat and human hepatocytes incubated with 0.5 mM EDB, and the level of DNA adduct formation in the human samples was about 40% of that in the rat hepatocytes. EDB concentration-dependent unscheduled DNA synthesis was demonstrated in isolated human hepatocytes. Concurrent treatment of the hepatocytes with diethylmaleate to deplete intracellular GSH inhibited EDB-induced unscheduled DNA synthesis. These results indicate that EDB alkylates DNA in human hepatocytes and that enzymatic repair of adducts may occur. The results of experiments done in rat and human systems using both purified GST enzymes and intact hepatocytes imply that the genotoxic pathway of EDB metabolism in rats and humans is similar.
Animals↗
Nuclear glutathione transferases which detoxify irradiated DNA.
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Animals↗
Decreased expression of the glutathione S-transferases alpha and pi genes in human renal cell carcinoma.
Using the polymerase chain reaction (PCR), a human kidney glutathione S-transferase (GST) alpha cDNA clone (GST alpha 12 K) was synthesized; it is identical to a known liver GST alpha cDNA clone except for one base change (G----A), indicating that an alpha class gene expressed in human kidney is similar to one expressed in human liver. Comparisons were made in the expression of GST alpha and GST pi between renal cell carcinoma and adjacent non-neoplastic tissue. Messenger RNA expression in 30 cases was determined by Northern blotting, and GST protein from nine of these cases was analyzed by HPLC. The GST alpha gene products were expressed at near-zero levels. The GST pi gene product was the predominant GST in tumors, but was decreased in absolute amount compared with control tissue, the tumor/control ratios for the GST pi gene obtained by Northern blots and HPLC analysis being 0.50 +/- 0.07 and 0.36 +/- 0.07 respectively. The resulting pattern in renal cell carcinoma therefore shows a predominance of GST pi. Since it is assumed that renal cell carcinoma derives from the proximal tubular epithelial cells which are high in GST alpha, this implies a dedifferentation in the GST expression pattern.
Blotting, Northern↗
The role of glutathione and glutathione transferases in chemical carcinogenesis.
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Animals↗
Changes in expression of mRNA coding for glutathione S-transferase subunits 1-2 and 7 in cultured rat hepatocytes.
mRNA hybridizing to probes for glutathione S-transferase (GST) subunits 1 and 2 (probe pGSTr 155) and subunit 7 (probe pGSTr 7) has been measured by Northern blot analysis in adult rat hepatocytes both in conventional monoculture and in co-culture with epithelial cells. In addition, several media conditions were used, namely with and without fetal calf serum (FCS) and with and without nicotinamide or dimethyl sulfoxide (DMSO). In monoculture, mRNA coding for subunits 1 and 2 was extensively reduced in the presence of FCS. In the absence of FCS, after an initial decrease, an increase of subunits 1 and 2 mRNA was noticed on day 6. When nicotinamide or DMSO was added to the medium, the GST subunits 1 and 2 mRNA level increased during the culture period. In co-culture, an initial reduction in levels of mRNA encoding subunits 1 and 2 was less marked and the values measured increased with co-culture time. Nicotinamide tended to reduce these mRNA levels, whereas DMSO increased them. In contrast, in conventional culture, mRNA encoding subunit 7 was expressed de novo and this induction was prevented by DMSO but not by nicotinamide. Similar results were obtained with co-culture.
Animals↗
Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.
Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.
Animals↗
Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes.
mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.
Animals↗
Control of expression of the human glutathione S-transferase pi gene differs from its rat orthologue.
We have examined regulation of the glutathione S-transferase pi gene by transient expression assay, and find that a fragment from 8 to 99 bp upstream of the cap site promotes transcription, but there is no evidence for any enhancer activity in a further 6 kb of flanking sequence. Analysis of this sequence by reference to a primate sequence database and Southern blotting revealed that as much as 5 kb of this flanking DNA were composed of repetitive insertion elements including an Alu and a LINE 1 repeat. The promoter fragment has been sequenced (Cowell et al (1988) Biochem. J. 255, 79-83) and contains a consensus AP1 binding site; in some cases, these have been associated with transcriptional induction by phorbol esters and ras oncogenes. We measured the steady state levels of glutathione S-transferase pi mRNA in human cell lines which were known to express ras oncogenes and compared them to human cell lines which have not been identified with ras activation. There was no correlation between expression of activated ras and expression of glutathione S-transferase pi mRNA. Treatment of HeLa cells, HepG2 cells and a small cell lung carcinoma line, GLC 8, with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate failed to alter the steady state levels of endogenous glutathione S-transferase pi mRNA. The differences between these results and those of similar studies on rat glutathione S-transferase subunit 7, a structural orthologue of glutathione S-transferase pi, are discussed.
Animals↗
Effect of phenobarbital on the expression of glutathione S-transferase isoenzymes in cultured rat hepatocytes.
Cultured adult rat hepatocytes were treated daily with 3.2 mM phenobarbital (PB) in order to study its effect on the expression of cytosolic glutathione S-transferase isoenzymes. Glutathione S-transferase (GST) activities, using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, were increased when PB was present in the culture medium. After purification and separation of GST on glutathione Sepharose 6 B and reversed-phase HPLC, respectively, it was observed in vitro that PB caused an increase in the relative amounts of subunits 1, 3 and 7 compared to subunits 2 and 4. Using Northern blot technique, elevated levels of GST subunit 1/2 and 7 mRNA were measured, after addition of PB to the cultures.
Animals↗
Single-step purification and h.p.l.c. analysis of glutathione transferase 8-8 in rat tissues.
GSSG selectively elutes two GSH transferases from a mixture of rat GSH transferases bound to a GSH-agarose affinity matrix. One is a form of GSH transferase 1-1 and the other is shown to be GSH transferase 8-8. By using tissues that lack this form of GSH transferase 1-1 (e.g. lung), GSH transferase 8-8 may thus be purified from cytosol in a single step. Quantitative analysis of the tissue distribution of GSH transferase 8-8 was obtained by h.p.l.c.
Animals↗
Purification and characterization of a labile rat glutathione transferase of the Mu class.
A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.
Animals↗
Glutathione transferases: a possible role in the detoxication and repair of DNA and lipid hydroperoxides.
Two types of GSH peroxidase occur in the cell both of which detoxify fatty acid hydroperoxides, thymine hydroperoxide and DNA hydroperoxides. One is a Se-dependent enzyme which also detoxifies H2O2. The other contains members of the GSH transferase supergene family. These non-selenium dependent GSH peroxidases do not detoxify H2O2 and have substrate specificities varying markedly with the isoenzyme. Of particular interest is GSH transferase 5*-5* an enzyme extracted from the nucleus with urea which has a relatively high activity towards DNA hydroperoxide. The possible role of these enzymes in the detoxication of lipid and DNA hydroperoxides is discussed and it is pointed out that they may be important participants in mechanism for the repair of free-radical damage.
Animals↗
Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety.
The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.
Animals↗
The structure of the human glutathione S-transferase pi gene.
The human glutathione S-transferase pi gene has been isolated from a cosmid library. The gene spans approximately 3 kb, is interrupted by six introns and the region around its 5' end has the high G + C and CpG content typical of an HTF (HpaII tiny fragment) island. In addition to a TATA box at position -28 relative to the transcription initiation site and two G + C boxes (GGGCGG), the promoter contains a sequence motif matching the phorbol ester- and ras-responsive element from the polyoma virus enhancer.
Amino Acid Sequence↗
Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus.
DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.
Animals↗